Treatment of tse infection

ABSTRACT

Transmissible spongiform encephalopathy (TSE) infection is treated by administration of an antibody that binds to prion dimer. A conjugate of a carrier and a fragment of a prion protein, optionally in oligomeric form and optionally having cyclic regions, is used to stimulate antibody production.

The invention relates to methods and compositions for the treatment ofinfection by transmissible spongiform encephalopathy (TSE) agents.

Transmissible spongiform encephalopathies (TSEs) are a group of fatalneurological diseases that include Creutzfeld-Jacob disease (CJD) andKuru in humans, bovine spongiform encephalopathy (BSE) in cattle andScrapie in sheep. TSEs are characterised by the conversion of a normalhost protein into a pathogenic protein within the brain tissue of aninfected animal. The pathogenic form of the protein is often referred toas a prion and is highly resistant to physical and chemical degradation.The prion is believed to be the transmissive agent through which the TSEdisease is passed on between animals.

There has been considerable public alarm in recent years over the risksassociated with consumption of meat products, and especially beef,potentially infected with BSE, the bovine form of TSE. Much of thisconcern is associated with the belief that the BSE prion when eaten by ahuman may in some cases cause the incurable human form of the disease,referred to as variant CJD (vCJD).

For those infected with TSE the prognosis is poor, with no effectivetherapies being available. Often the time from diagnosis to death isshort and uncomfortable both for the patient and for those around andcaring for the patient.

It is known to raise antibodies to fragments of prion proteins. Souan etal, Eur. J. Immunol. 2001: 31, pp 2338-2346 describe peptides thatinduce both T- and B-cell responses. However, no reduction in prionprotein tumour was achieved using the antibodies obtained.

A difficulty in raising antibodies to prion proteins is that peptidefragments are poor immunogens, and the antibodies obtained arefrequently of poor affinity. If pure disease-causing prion proteinitself is used this has the consequence that the resultant compositionis likely to contain disease-causing protein, and hence be unusable inthe clinic.

An anti-prion antibody, mAb 6H4 is also available commercially fromPrionics, Switzerland. This antibody can be used to detect prionprotein, typically using a second antibody conjugated to a detectablemarker, which second antibody binds to the first.

A difficulty that has been discovered by the present inventors is thatbinding of this antibody to equipment suspected of being contaminatedwith prion, or equipment that is suspected to be contaminated but whichhas been subjected to treatment intended to destroy the prion, does notcorrelate with infectivity. It has, for instance, been discovered by theinventors that prion-infected mouse brain homogenate, digested withprotease, run on SDS-PAGE, then probed with anti-prion antibody, shows anegative result, that is to say absence of antibody binding. Thismaterial nevertheless retains infectivity.

An object of the invention is to provide effective therapy, curativeand/or preventative, for those suffering from or at risk of infection orother disease caused by TSE agents.

A further object is to provide alternative and, in specific embodiments,improved production of antibodies that bind to prion proteins and can beused to treat prion disease.

Accordingly, the invention provides a method of treatment of TSEinfection, comprising administering an antibody that binds to a dimer ofa prion protein.

The antibody is preferably specific to the dimer, that is to say itbinds to the dimer but substantially does not bind to the monomer formof the prion, and may be obtained by methods described in detail below.

A further aspect of the invention provides a pharmaceutical composition,for treatment of TSE infection, comprising an antibody that binds to adimer of a prion protein.

An antibody of the invention is suitably obtained by immunising ananimal with an antigen that comprises prion protein or an analoguethereof, or a fragment of the protein or analogue, obtaining an extracttherefrom which contains antibodies, and isolating from said extractantibodies which bind prion dimer. Mouse, sheep, human and bovine (aswell as other) prion protein sequences are known and hence it isstraightforward to prepare a peptide that is a fragment of, say, atleast 7, preferably at least 10, more preferably at least 14 aminoacids. An analogue can be prepared by comparison of respective prionprotein sequences and synthesizing a composite with regions derived fromone or more prion protein source. Alternatively a synthetic sequence isprepared in which up to 2 amino acids in every 10 are substituted.

The antigen of the invention may comprise a mixture of peptides. Thesepeptides may comprise different regions from one or more prion proteinsequences, and/or the same regions from one or more prion proteinsequences, which same regions may contain intraspecies variation.

It is known that there is a high degree of homology between the prionprotein sequences of many mammals—both important functional residues andstructural elements are highly conserved. Accordingly, it is possible touse these conserved sequence regions, or fragments thereof, to preparethe immunising peptides of the present invention. These peptides cancomprise a variety of different regions from one or more prion proteinsequences. The high degree of prion protein sequence homology betweenspecies is set out in FIGS. 5 and 6.

In FIG. 5, the human prion protein precursor PrP^(c) was aligned withHamster, Sheep, Mule Deer, Elk, Mouse, Cow, Cat and Chicken homologousproteins using the ClustalW multiple alignment program (Higgins et al,1994). The sequences used for this alignment were taken from theSWISSPROT database and the database accession numbers of the sequencesused are as follows:— TABLE 1 SWISSPROT database accession numbers.Species Name SWISSPORT accession No. Human P04156 Mouse P04295 Hamster,P04273 Golden Sheep P23907 Cow P10279 Elk P79142 Mule Deer P47852 CatO18754 Chicken P27177 (representative of avian species)

Important structural features are marked above the aligned sequences ofFIG. 5 and numbering refers to the Human PrP^(c) sequence. Some of theimportant structural features of the sequences are as follows (terms inbrackets indicate how these features are marked on FIG. 5):—

-   1. The signal peptide is cleaved at position 22/23 to yield the    mature protein (Signal peptide)-   2. The N-terminus region is largely unstructured and flexible but    residues 37-53 can form an extended PPII helix forming a    hydroxylation site at Pro44 (Pro hydroxylation)-   3. The N-terminus also comprises a segment of 5/6 repeats which are    implicated in Copper binding (Repeat Region)-   4. Proteolysis can occur between Lys110 and His 111 (Cleavage)-   5. There is a short hydrophobic residue region (Hydrophobic region)    believed to be a transmembrane segment.-   6. The C-terminus is characterised by a bundle of three α-helices (a    1-3) and two β-sheets (b1-2)-   7. A di-sulphide bridge occurs between residues Cys179 and Cys214    (-SS-)-   8. Asparagine residues Asn181 and Asn197 are available for    N-glycosylation (Carbohydrate groups; the conserved motif being    Asn-X-Thr (Asn-Gly).-   9. Residue Ser231 provides the residue where the GPI (glycolipid)    anchor moiety is attached (GPI anchor).

On inspection of the multiple alignment of FIG. 5, the followingfunctionally important residues were absolutely conserved in themammalian species shown:

-   -   Signal Peptide cleavage site (C23-K24)    -   Pro44 hydroxylation site,    -   Proteolytic cleavage site (KI 10-H 111)    -   Arg-glycosylation sites (R181, R197)    -   Disulphide bridge (C179, C214)    -   GPI anchor (S231)

Regions which differ tend to be in the N-terminal sequence and some ofthe structural elements (e.g. α-helices), although the β-sheets and thehydrophobic region are totally conserved.

FIG. 6 shows the percent identifies derived from the alignment ofindividual pairs of protein sequences. Again, this figure demonstratesthe high level of identity between mammalian species of the prionprotein amino acid sequence. Preferably, peptides for use in methods andcompositions of the invention are prepared from fragments of theidentified conserved regions.

One way to make an antibody selective for prion dimer is to immunise ananimal and extract serum. This is run on a prion dimer column toidentify antibodies that bind the dimer. These antibodies are thentested for cross-reactivity with prion monomer, with cross-reactingantibodies removed. The removal can be effected using a column loadedwith prion monomer, the antibodies that emerge therefrom then beingtested for absence of cross-reactivity.

Isolated prion dimer forms a further embodiment of the invention. Thisisolated material can be obtained from the column in the methoddescribed immediately above or simply by cutting out a portion of theSDS-PAGE gel used to resolve prion dimers. Alternatively, otherseparation techniques can be used to extract the prion dimer fromhomogenised prion-infected mouse brain. Antibodies that bind to theprion monomer and which are cross reactive can be used to confirm thatthe material thus obtained is prion dimer and not other protein of thesame molecular weight.

In a preferred embodiment of the invention, the antibody is obtained byimmunising an animal with a peptide that is or comprises a fragment ofprion protein optionally supplemented by a cysteine residue at one orboth ends, in specific examples one selected from SEQ ID NO:s 1 to 8.The use of cysteine residues (reference to the sequence or fragment isintended to include reference to the sequence or fragment with 0, 1 or 2cysteine residues at its ends) offers the advantage that the peptidesmay form oligomers due to cysteine-cysteine bonds. As a result theimmunizing peptides include linear monomers, linear dimers, cyclicmonomers, cyclic dimers and other oligomeric forms with repeated prionpeptide sequences and cyclic regions. These peptides allow a wider rangeof immunising antigens to be presented to the animal. Cyclic forms arepresented which may more closely mimic the natural form of thedisease-forming agent. Dimeric forms, some of them cyclic, are presentedwhich again may more closely mimic the dimeric forms of thedisease-causing agent. The invention hence extends to all fragments ofall prion sequences described in FIG. 5, wherein the fragments are atleast 7 amino acids in length and have cysteine residues at one or bothends.

The peptide may be introduced into the animal via various routes ofadministration. In a preferred embodiment, the peptide is injected intothe peritoneum, a site which is (a) surgically easily accessible, (b)allows a large volume of liquid to be injected at any one time, and (c)allows rapid absorption of the peptide into the bloodstream.

In use, the antibody can be delivered to the systemic circulation.Binding of the antibody to prion dimer results in removal anddestruction of that dimer and reduction of infection. It is believedthat prion dimer originating in the brain will diffuse across the bloodbrain barrier and that the presence of antibodies on the systemic sideof the barrier will create a concentration gradient as dimer is moppedup after crossing the barrier, this helping to reduce infection anddisease.

It is known to conjugate antibodies to transport vectors so thatantibodies can cross the blood brain barrier. For example, an antibodycan be coupled via avidin/biotin to the OX 26 monoclonal antibody, whichlatter antibody acts as a transport vector for crossing the blood brainbarrier. Further details are described in Bickel et al, Advanced DrugDiscovery Reviews 46 (2001) 247-279. A further embodiment of theinvention hence lies in an antibody adapted, for example using thetechnology reviewed by Bickel et al, to cross the blood brain barrier.

A further preferred embodiment of the invention lies in enhancing theimmune response to the antigen by use of a carrier. Hence, antibodiesare obtained by immunising an animal with an antigen, wherein theantigen comprises a peptide of the invention that is, or comprises afragment of, a prion protein or an analogue of a prion protein, andwherein the antigen further comprises a carrier covalently linked to thepeptide, optionally via a linker. This has the advantage of improvedstimulation of antibody production. The carrier can be selected from awide range of immunogenic carrier substances, e.g. proteins, heat shockproteins, toxoids, including such examples as bacterial proteins andbacterial toxoids, particularly mycobacterial proteins, pertussisproteins and toxoids, diphtheria proteins and toxoids. De Silva et al.,Bioconjug Chem 1999 May-June; 10(3); 496-501 describe use of PPD as animmunogenic carrier, and heat shock proteins as carriers are describedin Lussow et al, Immunol 1991 October: 21(10); 2297-2302. The animal canbe a source of antibodies, in which case antibodies can be obtained fromthat animal and those that bind to prion dimer identified. The animalcan be one being or to be treated. The invention hence also providesmethods of treating TSE and/or immunizing an animal against TSEinfection, comprising administering the peptide of the invention and/orthe antigen of the invention.

It is, in addition, preferred to sensitise the animal to the carrier,either prior to or at the same time as administering theantigen/carrier. Thus, in a particular method described in the examples,antibody production comprises administrating a priming antigen thatstimulates an immune response to the carrier. Again, an advantage isimproved antibody production. The priming antigen may be carrier on itsown or a fragment of the carrier.

In a specific example, the carrier is a Mycobacterial protein and thepriming antigen is administered by administering BCG vaccine. Furtherdetails of this approach are described for example in Lussow et al,Immunol 1991 October: 21(10); 2297-2302.

Still further aspects of the invention lie in isolated peptides andantigens of the invention and their uses.

The term transmissible spongiform encephalopathy (TSE) agent is intendedto encompass all neurological diseases that are apparently transmittedvia a pathogenic prion protein intermediate. Such TSEs typically includethe human diseases Creutzfeld-Jacob disease (CJD), variantCreutzfeld-Jacob disease (vCJD), Kuru, fatal familial insomnia andGerstmann-Straussler-Scheinker syndrome. Non-human TSEs include bovinespongiform encephalopathy (BSE), scrapie, feline spongiformencephalopathy, chronic wasting disease, and transmissible minkencephalopathy. Given that vCJD is currently understood to be a humanform of BSE, it is apparent that certain TSE agents are capable ofcrossing the species barrier and that novel TSEs from non-bovine sourcescould become evident in future. Reference to TSE infection refers alsoto prion disease.

The antibodies and/or peptides of the invention can be used in aneffective therapy, curative and/or preventative, for clearing all orpart of the UK herd of scrapie (sheep). In sheep, there are often flockswhere up to 20% of the flock catches scrapie. It would therefore bebeneficial to vaccinate isolated flocks known to be at risk, and/or totreat infected sheep with antibodies that bind to prion protein. Theantibodies and/or peptides of the invention may similarly be used toclear all or part of the UK herd of BSE (cattle).

A further aspect of the invention lies in the use of the antibodiesand/or peptides to treat humans presenting with the signs of, oridentified as at risk of, CJD or new variant CJD.

In another embodiment, antibodies and/or peptides of the invention allowearly diagnosis of a TSE infection based on tissue removed from, forexample, the appendix, tonsils or through a lumbar puncture. With earlydiagnosis, the prospects of success in treatment, or at least prolonginglife by treatment, are increased.

In yet another embodiment, antibodies and/or peptides of the inventionare used to provide a wide-spread vaccination program in animals. It isenvisaged that such a vaccination program could be carried out withoutprior diagnosis or testing.

The antibodies of the invention are optionally monoclonal antibodiesthat bind to prion proteins, preferably specifically to prion dimers,and can be used to treat prion disease.

As set out in more detail in the examples, prion strain 301V, a mousepassaged isolate, derived from a Holstein-Fresian cow terminally illwith BSE is used as an example of a prion strain. Infection is known tobe produced by intracerebral inoculation and the incubation periodrequired for the onset of clinical symptoms is remarkably uniform. Thatis to say, providing that the dose of infectious agent is sufficientthen the classic signs of disease will appear at a defined timepost-inoculation (in VM mice this is 120 days). For obvious reasonsno-one has tested these properties on humans, however, the mousebioassay is regarded as the closest available model and therefore a goodindicator of BSE infection in man.

The methods and compositions of specific embodiments of the inventionsare described in more detail below and are illustrated by theaccompanying drawings in which:—

FIGS. 1 to 4 show blots of BSE (301V)-infected mouse brain homogenate,to illustrate binding of antibodies of the invention to prion dimer.

FIG. 5 shows the multiple alignment of selected mammalian and avianPrP^(c) proteins.

FIG. 6 shows the pair sequence distances of selected mammalian and avianPrP^(c) protein sequences.

EXAMPLES Dimer Detection in Digested Mouse Brain

BSE (301V)-infected mouse brain homogenate was digested at neutral pHand 60° C. for 30 minutes with protease. Total protein digests were runon SDS-PAGE and transferred by Western blotting to nitro-cellulosemembranes. These were cut into strips and probed with CAMR anti-prionantibodies (produced in rabbits). A second generic antibody (goatanti-rabbit) was conjugated to horseradish peroxidase and used withdetection by TMB colorimetric substrate.

At the time, the expected result was that the results were the same asthe control blot (number 7) using the anti-prion antibody mAb 6H4 (fromPrionics, Switzerland). In this control blot, there is seen the typicalthree-banded pattern (glycosylation states) for protease-digestedinfectious-conformation prion protein (PrPSc).

However, the blots in this example did not show this pattern. Blot 1uses a polyclonal antibody raised against a PPD-conjugated peptidecorresponding to an N-terminal region of the prion molecule. Nothing isseen in the lanes. This section of the protein is susceptible toproteolysis, so it is not surprising to see nothing in the lanes (2 &3)—see FIG. 1, blot 1 on left hand side. Lane 1 is a molecular weightmarker.

Blot 2 has a second antibody raised against a peptide sequence furtherinto the prion molecule. This shows at least 9 bands of varyingintensity, approximately equidistant, at a molecular weightcorresponding to a prion dimer with a range of glycosylation states—seeblot 2 on FIG. 1.

Blot 3 antibody shows similar profile; blot 4 is also shown but itsresults are too poor quality to draw any conclusions—see blots 3 and 4on FIG. 1.

Blots 5 and 6, shown on FIG. 2 with the control blot 7, again show themulti-banded pattern.

Dimer Detection in Digested Mouse Brain

The above example was repeated, and the results shown in FIGS. 3 and 4.

Blot 1 shows molecular weight markers in lanes 1 and 5. Lane 2 isrecombinant murine PrP showing recombinant murine PrP oligomers. Lane 3shows lack of antibody response to protease-digested infectious mousebrain homogenate. Lane 4 is the antibody response in the undigestedcontrol.

Blot 2 is as above but shows the previous banding pattern in theprotease digested sample.

Blot 3 shows the antibody 3 response. Here there is some response torecombinant murine PrP (lane 2). Lane 3 shows not only the multiple(dimeric PrP) banding pattern, but also some monomeric PrP response.

Blot 7 is the 6H4 mAb antibody control. Here there is good detection ofrecombinant murine PrP oligomers (lane 2). Lane 3 shows the heavilydiglycosylated form of limitedly protease-treated PrPSc, plus the moreminor monoglycosylated and non-glycosylated forms typical of BSE (301V)strain. No dimer detection is apparent.

Preparation of Antibodies Including Dimer Preferential Antibody

In the examples, we have used 6 polyclonal antibodies. Of these, threedetect the dimer alone and do not bind the monomer, whereas onecross-reacts with both the monomer and the dimer.

The polyclonal sera were produced by immunisation of rabbits withsynthesised prion mimetic peptides. These peptides were designed basedon regions of high homology between human, mouse and bovine prionprotein amino acid sequences.

SEQ ID No:s 4, 5, 7 and 8 produced the dimer-reactive antibodies.

The peptides were synthesised with a cysteine at both ends (see above)and with a cysteine at one end only. This method was used in order topresent both the linear form and a loop structure of the antigen on thesurface of the carrier protein.

The peptides were synthesised commercially and coupled to the carrierprotein PPD (purified protein derivative), derived from an attenuatedstrain of the bacterium Mycobacterium bovis, which is lyophilised andused to conjugate to the peptide via a linker.

Anti-prion polyclonal antibodies were produced as follows:

A sample of pre-immune sera (−1 ml) was collected from each of a groupof Dutch rabbits. The rabbits were injected with reconstitutedfreeze-dried Bacillus Calmette-Guerin (BCG) vaccine for intradermal use.A dose of 0.1 ml of reconstituted BCG vaccine was given in two sites inthe scruff of the neck of the rabbit. After 4 weeks, 0.6 mg of eachpeptide-PPD conjugate was measured (0.3 mg of each of the 1 cysteine and2 cysteine versions) and dissolved in 1 ml of sterile 0.9% saline.

An equal volume of incomplete Freunds adjuvant was added and 0.75 mlaliquots of the resulting emulsion were injected intra-muscularly intoeach hind limb, and 0.25 ml aliquots into two sites in the scruff of theneck per rabbit. After 4 weeks a boost injection was given comprising ofthe peptide-PPD conjugates prepared as in step 3 and 4. The boostinjections consist of four 0.25 ml injections into the scruff of theneck of each rabbit. 7-14 days after the first boost injections, 4 mltest bleeds were taken, the sera was assessed by ELISA for antibodytitre. A second boost injection was given 4-6 weeks after the first.

A third boost injection given 4-6 weeks later. A 4 ml test bleed wastaken 6-8 weeks after the third boost injection and antibody titresdetermined by ELISA. A fourth boost injection was then given.

A 4 ml test bleed was taken 7-14 days after the fourth boost injectionand antibody titre determined by ELISA. Terminal exsanguination wascarried out and blood collected. The serum was separated bycentrifugation and stored at minus 20° C.

Analysis of antibody titre was achieved using ELISA. The immunoassayplate was coated with the same peptides conjugated to a differentcarrier protein (KLH) in order to differentiate the response to thepeptide from the response to the carrier protein.

Three of the antibodies produced by immunisation of the syntheticpeptide sequences described bind preferentially to the dimer form of themolecule.

Immunisation Protocol Using PPD Coupled Prion-Derived Peptides for theTreatment of Prion Infection in Mice.

VM mice were inoculated intra-cerebrally with 20 ml of a 0.1% (w/v)suspension of BSE (301 V) infective mouse brain homogenate. After twoweeks the mice were inoculated with BCG to prime the immune response toinoculated peptide conjugates. After a further 2 weeks the mice wereinoculated with 100 ml of PPD-peptide conjugate mixed 1:1 withincomplete Freund's adjuvant or suitable controls. The peptides of theconjugates were selected from SEQ ID No:s 1-8, and corresponded asfollows:— Peptide 1 = SEQ ID No: 5 Peptide 2 = SEQ ID No 6 Peptide 3 =SEQ ID No 8

Three further inoculations at the same concentration of conjugate wereadministered at 2-weekly intervals. Mice were observed for clinicalsigns of the progression of prion disease over the next 2-6 months.Results of the experiment after 138 days are summarised in Table 2.TABLE 2 Use of PPD coupled prion-derived peptides for the treatment ofprion infection in mice. No. of First Mouse Last Mouse Mean SD TreatmentMice culled on day:- culled on day:- (days) (days) BCG only  9 123 133129.7 5.0 BCG + 10 128 128 128.0 0.0 PPD only BCG + 10 133 133 133.0 0.0PPD- peptide 1 BCG +  9 133 138 135.4 2.0 PPD- (2 mice still peptide 2alive on day 138) BCG + 10 128 132 131.2 1.2 PPD- peptide 3

The results of this experiment indicate that immunisation with each ofthese PPD-peptides was successful in increasing the mean survival timeof mice pre-exposed to TSE. Immunisation with PPD-peptide 1 andPPD-peptide 2 provided particularly notable increases in the meansurvival time of mice preexposed to TSE. A statistical analysis of theresults for these two peptides at the end of 138 days is provided below.

In interpreting the results of the above experiment, it should berecognised that the experiment represents an extremely rigorous test ofthe effectiveness of the peptides for the treatment of prion infectionin mice, as the mice have been pre-exposed to TSE. Peptides of theinvention thus are shown to increase survival time of mice pre-exposedto TSE and are suitable candidates for use in curative therapies.

The peptides are also suitable for use in prophylactic therapy, i.e. byexposing the animals to a peptide-carrier conjugate before exposure tothe infectious agent.

Statistical Analysis of the Effect of PPD-Peptide 1 and PPD-Peptide 2 onthe Mean Survival Time of Mice Pre-Exposed to TSE

One-way ANOVA: Group 2 (PPD-peptide 2) versus Group 8 (control-BCG only)Analysis of Variance for Group 4 Source DF SS MS F P Group 8 1 26.88926.889 35.29 0.001 Error 7 5.333 0.762 Total 8 32.222 Individual 95% CIsFor Mean Based on Pooled StDev Level N Mean StDev-------+---------+---------+--------- 123 3 133.000 0.000(------*-------) 133 6 136.667 1.033 (----*----)-------+---------+---------+--------- Pooled StDev = 0.873 132.8 134.4136.0

Two-Sample T-Test and Cl: Group 8 (control-BCG only), Group 2(PPD-peptide 2) Two-sample T for Group 8 vs Group 2 N Mean StDev SE MeanGroup 8 9 129.67 5.00 1.7 Group 2 9 135.44 2.01 0.67 Difference = muGroup 8 − mu Group 2 Estimate for difference: −5.78 95% CI fordifference: (−9.78, −1.78) T-Test of difference = 0 (vs not =): T-Value= −3.22 P-Value = 0.009 DF = 10

Mann-Whitney Test and Cl: Group 8 (control-BCG only), Group 2(PPD-peptide 2) Group 8 N = 9 Median = 133.00 Group 2 N = 9 Median =136.00 Point estimate for ETA1-ETA2 is  −3.00 99.2 Percent CI forETA1-ETA2 is (−13.00, −0.00) W = 54.0 Test of ETA1 = ETA2 vs ETA1 < ETA2is significant at 0.0031 The test is significant at 0.0016 (adjusted forties)

Statistically, the observed difference between the control group and thegroup immunised with PPD-peptide 2 has been shown to be stronglysignificant.

Mann-Whitney Test and CI: Group 8 (control-BCG only), Group I(PPD-peptide 1) Group 8 N =  9 Median = 133.00 Group 1 N = 10 Median =133.00 Point estimate for ETA1-ETA2 is  −0.00 95.5 Percent CI forETA1-ETA2 is (−10.00, 0.00) W = 75.0 Test of ETA1 = ETA2 vs ETA1 < ETA2is significant at 0.1182 The test is significant at 0.0628 (adjusted forties) Cannot reject at alpha = 0.05

Two-Sample T-Test and Cl: Group 8 (control-BCG only), Group I(PPD-peptide 1) Two-sample T for control vs PPD-peptide 1 N Mean StDevSE Mean Group 8 9 129.67 5.00 1.7 Group 1 10 133.000 0.471 0.15Difference = mu Group 8 − mu Group 1 Estimate for difference: −3.33 95%CI for difference: (−7.19, 0.53) T-Test of difference = 0 (vs not =):T-Value = −1.99 P-Value = 0.082 DF = 8

Statistically, the observed difference between the control group and thegroup immunised with PPD-peptide 1 is significant.

Immunisation of Human Subjects Infected with Prion Disease

Peptides are conjugated to PPD or BCG carrier proteins essentially asoutlined above. The conjugate is formulated in such a way as to make itsuitable for immunisation into clinical patients using methods andcompositions known to those familiar with the art.

An immunisation protocol based on an initial immunisation with conjugatefollowed by two 2-weekly injections at 50% concentration followed by anadditional injections at 2, 3 and 4 months is suitable for therapeutictreatment of individuals.

Production of Rabbit Polyclonal Antibodies and Mouse MonoclonalAntibodies

The production of polyclonal antibodies in rabbits is outlined below:

Dutch rabbits (approximately 2.5 kg) were allowed to settle in newaccommodation for approximately two weeks and a sample of pre-immuneserum (˜1 ml) collected from each. Inject each rabbit with reconstitutedfreeze-dried Bacillus Calmette-Guerin (BCG) vaccine for intradermal use(Statens Seruminsitut, Denmark). Dose with 0.1 ml of reconstituted BCGvaccine in each of two sites in the scruff of the neck of the rabbit.Leave all rabbits for 4-6 weeks before first injection of peptide-PPDconjugates.

-   -   Combine each of the peptide-PPD conjugates (1 ml) supplied with        an equal volume of incomplete Freund's adjuvant and inject 0.75        ml aliquots of each resulting emulsion intramuscularly into each        hind limb and 0.25 ml aliquots into two sites in the scruff of        the neck per rabbit. Leave for 4-6 weeks.    -   Give first boost injection 4-6 weeks following first peptide-PPD        conjugate incomplete Freund's adjuvant inoculation. Boost        injections to comprise only four 0.25 ml injections into the        scruff of each rabbit. Second boost to take place in 4-6 weeks        time.    -   Take test bleed (−4 ml) 7-14 days following first boost        injection.    -   Give second boost injection 4-6 weeks following first boost        injection. Boost injection again to comprise only four 0.25 ml        injections into the scruff of each rabbit. Third boost to take        place in 4-6 weeks time.    -   Give third boost injection 4-6 weeks following second boost        injection. Boost injection again to comprise only four 0.25 ml        injections into the scruff of each rabbit.    -   Take second test bleed (−4 ml) 6-7 weeks following third boost        injection and give fourth boost injection in approximately a        further weeks time.    -   Take third test bleed (−4 ml) 7-14 days following fourth boost        injection. Subject to serum analysis results, carry out terminal        exsanguination within the next three days.

The immunisation protocol for the production of antibodies in mice issimilar to that described above:—

-   1. Injection with reconstituted freeze dried Bacillus Calmette    Guerin (BCG) Intradermal (day 0) 25 ml per mouse-   2. Peptide—PPD conjugate in Freunds Incomplete Adjuvant ip (day 28)    80 mg/mouse-   3. Repeat as DAY 28 (day 42) 40 mg/mouse-   4. Repeat as DAY 28 (day 49) 40 mg/mouse-   5. Tail vein test bleed (day 56)-   6. Peptide—PPD conjugate (must be without adjuvant) iv (day 63)-   7. Fusion on selected mice (day 66)

The immune response is assessed by ELISA using either plates coated withfree peptide or peptide-KLH conjugate using doubling dilution toestimate end-point. Titres of between 1:51,200 and 1:102,000 aretypically obtained for the immunised animals on ELISA plates coated withfree peptide.

For the generation of monoclonal antibody cell lines the spleen of theimmunised mouse is removed and fused with myeloma cell lines usingstandard methods (Antibodies; A laboratory Manual, Ed Harlow and DavidLane, Chapter 6, p196-224. (Cold Spring Harbour Laboratory, 1988)). Anexample of a suitable method is provided below, although those familiarwith the art will recognise that other similar protocols could be usedfor generation of antibody producing cell lines.

Preparing Splenocytes for Fusions

-   1. Sacrifice mouse. Aseptically remove spleen and place on a tissue    culture dish containing 10 ml of serum free medium. Trim off and    discard contaminating tissue from spleen.-   2. Tease apart the spleen using 19 gauge needles on 1 ml syringes.    Continue to tease until most of the cells have been released and the    spleen has been tom into fine parts. Disrupt any cell clumps by    pipetting. Transfer the cells and medium into sterile centrifuge    tube.-   3. Wash the tissue culture plate and tissue clumps with 10 ml serum    free medium (prewarmed to 37° C.) and combine with the first 10 ml    in the tube.-   4. Allow the cell suspension to sit for approx. 2 min. Carefully    remove the supernatant from the sediment and transfer to a fresh    centrifuge tube.    Fusion Technique

Prior to the fusion the myeloma cells that will serve as fusion partnersmust be removed from frozen stocks and grown.

-   1. Melt a vial of 0.3 g of PEG in a 50° C. water bath. Add 0.7 ml of    medium without serum and transfer to a 37° C. water bath.-   2. Centrifuge the spleen cells from the immunised animal at 400 g    for 5 minutes. Simultaneously centrifuge 20 ml of myeloma cells.    Resuspend both cell pellets in 5 ml of medium without serum.-   3. Combine the two cell suspensions and transfer to a 15 ml    round-bottomed centrifuge tube. Centrifuge for 5 min at 400 g.    Carefully remove all medium.-   4. Add 0.2 ml of PEG solution. Suspend the cells by lightly tapping    the tube.-   5. Centrifuge for 5 min at 400 g. Add 5 ml of medium without serum    to disperse the pellet. Flick the tube, if necessary, to resuspend    the cells. Do not pipet the cells. Then add 5 ml of medium with 20%    Foetal calf serum.-   6. Centrifuge for 5 min at 400 g. Remove the supernatant and    resuspend the cells in 10 ml of medium supplemented with 20% foetal    calf serum, 1×OPI and 1×HAT. Add the cells to 200 ml of medium    supplemented with 20% foetal calf serum and 1×HAT.-   7. Dispense 100 ml of cells into the wells of microtitre plates.    Place at 37° C. in a CO₂ incubator.-   8. Cells are fed by the addition of 100 ml of fresh medium    supplemented with 20% Foetal calf serum containing HAT after 4-5    days.

OPI is a media supplement used to help support the growth of cellsplated at low cell densities. It is a solution of oxaloacetate, pyruvateand insulin.

HAT—hypoxanthine, aminopterin and thymidine (drug selection media).Other commonly used drug selection methods are AH (azaserine andhypoxanthine).

Single Cell Cloning

After a positive tissue culture supernatant has been identified, thenext step is to clone the antibody producing cell.

-   -   Using a multiwell pipettor, add 50 ml of medium with 20% FCS and        2×OPI to each well of a 96 well plate.    -   Remove 100 ml of hybridoma cell suspension and transfer to top        left-hand well. Mix by pipetting.    -   Do doubling dilutions down the left-hand column of the plate. (8        wells, 7 dilution steps). Discard tip.    -   Do doubling dilutions across the plate using a 8-well        multichannel pipette. Clones should be visible after a few days        and ready to screen after 7-10 days. Select the best wells, grow        up and repeat the cloning procedure.        Generation of Humanized Anti-Prion Dimer Antibodies by CDR        Grafting

The therapeutic potential of monoclonal antibodies that recogniseprion-dimers may be significantly advanced by grafting the variableregions of the mouse antibodies onto the human variable domain by aprocess called CDR grafting (as described in Antibody engineering; apractical approach Eds McCafferty, J., Hoogenboom, H. R. and Chiswell,D. J. Oxford Univeristy Press 1996 and references therein). This methodreduces the immunogenicity of the therapeutic antibody preparation byreplacing much of the mouse antibody with the equivalent human protein.The method is briefly outlined below:

cDNAs encoding mouse monoclonals, produced and characterised asdescribed above, are amplified by PCR using specific oligonucleotideprimers designed to the heavy and light chain variable regions. Thesevariable regions are cloned onto the constant regions of humanantibodies to generate a chimeric antibody using methods known to thosefamiliar with the art (as outlined in references including Antibodyengineering; a practical approach Eds McCafferty, J., Hoogenboom, H. R.and Chiswell, D. J. Oxford Univeristy Press 1996). These chimericantibodies are expressed recombinantly in either mammalian cells (e.g.Chinese hamster ovary cells; CHO) or E. coli.

Recombinant Expression of Antibody Fragments in E. coli.

For recombinant expression in E. coli the variable region eitheramplified from the original mouse monoclonal antibody cell line or fromthe humanised chimeric antibody is expressed either as an scFV fragmentor a Fab fragment expressed into the cytoplasm of suitable E. colistrains (eg trxB mutants) or periplasmically using methods known tothose familiar with the art and as outlined in Antibody engineering; apractical approach Eds McCafferty, J., Hoogenboom, H. R. and Chiswell,D. J. Oxford Univeristy Press 1996 and subsequent references.

To facilitate the purification of scFV fragments the variable regionsare cloned into an expression vector containing a 6-histidine tag ateither the N or C terminus. The addition of the tag has no effect on therecognition of the prion-dimer by the scFV or on function. Forexpression of the protein the clone in a trxB mutant E. coli strain(e.g. AD494 or BL21trxB; Novagen) is inoculated into 100 ml of LB withsuitable antibiotic and grown overnight at 30° C. The culture is diluted1:50 into fresh media and grown at 30° C. to an OD_(600nm) of 0.6-1. Theculture is induced by addition of IPTG or other inducer appropriate tothe expression system and the culture grown at 25° C. for a further 34hours. Cell material is isolated by centrifugation.

Purification of Antibodies.

Antibody fragments are purified from either hybridoma supernatant, fromCHO cells transfected with humanised IgG or from recombinant E. colicultures using standard methods known to those familiar with the art. Inbrief, monoclonal antibodies are purified by affinity chromatography onProtein G or Protein A—sepharose columns following an (optional)ammonium sulphate precipitation from crude culture supernatant.Typically the crude supernatant from a hybridoma or CHO cell line isammonium sulphate precipitated by addition of ammonium sulphate to 40%saturation and incubated for at least 1 hour at 4° C. The precipitate iscollected by centrifugation at 15000 g for 30 min at 4° C. The pellet isresuspended in phosphate buffered saline (PBS) at a final concentrationof approximately 2 mg/ml and loaded onto a protein-G-sepharose orprotein-A sepharose column (Pharmacia) equilibrated in phosphatebuffered saline according to manufacturers instructions. Purifiedantibody is eluted using 100 mM glycine pH 2.8 and collected directlyinto a concentrated phosphate buffer at high pH. The purified protein isdialysed extensively against PBS.

Expression and Purification of Recombinant Antibody Fragments in E.coli.

Purification of the recombinant scFV fragment from E. coli is carriedout using standard methods. In the case of the his-tagged scFv fragmentoutlined in example 5 the cell pellet from the expression culture islysed using either sonication or by proprietary detergent lysis (e.g.Bugbuster; Novagen) and clarified by centrifugation. The supernatantfraction is applied to an immobilised metal ion affinity chelate (IMAC)column (Pharmacia) loaded with Cu or Ni in 50 mM HEPES 150 mM NaCl pH7.4or similar buffer. Following washing to remove non-specifically boundproteins the scFV is eluted using a gradient of 0-500 mM imidazole inthe same buffer. The imidazole is removed by dialysis prior to storage.Other standard protein purification methods are also suitable for theisolation of recombinant antibody fragments from E. coli.

Formulation of Therapeutic Antibody Preparation for the Treatment ofPrion Disease

Purified monoclonal antibodies or recombinant chimeric antibodies andfragments thereof are prepared as described above and shown to be freeof endotoxin contamination. Antibody is formulated either with orwithout suitable carrier proteins (e.g. serum albumin), with or without0.9% sodium chloride and in the presence or absence of suitablestabilising agents such as dextrose, sorbitol, sucrose or manitol. Theantibody should be formulated at a concentration of 10 mg/ml.

Treatment of Prion Disease with Therapeutic Antibodies

For therapeutic application of the anti-prion dimer monoclonal antibodysimilar protocols can be used to those used for the treatment oflymphoma or other cancers with monoclonal antibodies. An example of asuitable dosing schedule is to prepare the formulated antibody as aninfusion at a concentration of 1 mg/ml (final concentration) in 0.9%sodium chloride with 5% dextrose in water. The infusion is applied as aslow i.v. infusion over several hours. The concentration of antibody tobe used is up to 400 mg/m² of body surface area and the dosage isrepeated once weekly over eight weeks. Alternative dosing schedules suchas 600 mg/m² once weekly for 4 weeks or other schedules known to thosefamiliar with the art would also be suitable for administration of theantibody.

Improving the Uptake of Therapeutic Antibody Fragments Across theBlood-Brain Barrier

The effectiveness of the therapeutic application of anti-prion dimerantibodies depends on the ability of the antibody to reach the centralnervous system (CNS). A number of the available methods to promoteantibody access to the CNS, when used in conjunction with theinoculation of therapeutic antibody, may provide enhanced effectivenessover inoculation alone. Specific examples of such processes would be; 1)the conjugation of the therapeutic antibody to an anti-transferrinreceptor antibody using standard methods outlined in: Lee H J.Engelhardt B. Lesley J. Bickel U. Pardridge W M. (2000) “Targeting ratanti-mouse transferrin receptor monoclonal antibodies throughblood-brain barrier in mouse J Pharmacol Exp Ther. 292: 1048-52 andsimilar papers 2) the use of the drug Cereport (Alkermes Cambridge,Mass.) to lower the blood brain barrier following inoculation 3)conjugation of the antibody to an agent such as tetanus toxin receptorbinding domain (Hc) for delivery via retrograde transport, 4) directinoculation of recombinant antibody producing cells (such as glial, orSchwan cells) into the CNS.

The invention thus provides treatment of TSE infection and antibodiestherefor.

1-41. (canceled)
 42. A method of treatment of TSE infection, comprisingadministering an antibody that binds to a prion, wherein the prioncomprises a PrP^(Sc) prion dimer that is infectious in animals.
 43. Amethod according to claim 42, wherein the antibody is specific toPrP^(Sc) prion dimer.
 44. A method according to claim 42, wherein theantibody is obtained by immunising an animal with a prion protein or ananalogue thereof, or with a fragment of the protein or the analogue,obtaining an extract therefrom which contains antibodies, and isolatingfrom said extract antibodies that bind to a prion, wherein the prioncomprises a PrP^(Sc) prion dimer that is infectious in animals.
 45. Amethod according to claim 42, wherein the antibody is obtained byimmunising an animal with a peptide that comprises a fragment of prionprotein.
 46. A method according to claim 45, wherein the peptide isselected from SEQ ID NO:s 1 to 8, optionally supplemented by a cysteineresidue at one or both ends.
 47. A method according to claim 45, whereinthe peptide is in a linear conformation, optionally a linear dimer, or acyclic conformation, optionally a cyclic dimer.
 48. A method accordingto claims 45, wherein the peptide comprises a repeated fragment of aprion protein.
 49. A method according to claim 42, for treatment of adisease selected from the group consisting of Creutzfeld-Jacob disease;variant Creutzfeld-Jacob disease; Kuru; fatal familial insomnia;Gerstmann-Straussler-Scheinker syndrome; bovine spongiformencephalopathy; scrapie; feline spongiform encephalopathy; chronicwasting disease; and transmissible mink encephalopathy.
 50. A methodaccording to claim 42, wherein the antibody is a monoclonal antibody.51. A pharmaceutical composition for treatment of TSE infection,comprising an antibody that binds to a prion, wherein the prioncomprises a PrP^(Sc) prion dimer that is infectious in animals.
 52. Acomposition according to claim 51, wherein the antibody is specific toPrP^(Sc) prion dimer.
 53. A composition according to claim 51, whereinthe antibody is obtained by immunising an animal with a prion dimer,obtaining an extract therefrom which contains antibodies, and isolatingfrom said extract antibodies that bind a prion, wherein the prioncomprises a PrP^(Sc) prion dimer that is infectious in animals.
 54. Acomposition according to claim 51, wherein the antibody is obtained byimmunising an animal with a peptide that comprises a fragment of prionprotein.
 55. A composition according to claim 54, wherein the peptide isselected from SEQ ID NO:s 1 to 8, optionally supplemented by a cysteineresidue at one or both ends.
 56. A composition according to claim 54,wherein the peptide is in a linear conformation, optionally a lineardimer.
 57. A composition according to claim 54, wherein the peptide isin a cyclic conformation, optionally a cyclic dimer.
 58. A compositionaccording to claim 54, wherein the peptide comprises a repeated fragmentof a prion protein.
 59. A composition according to claim 51, fortreatment of a disease selected from the group consisting ofCreutzfeld-Jacob disease; variant Creutzfeld-Jacob disease; Kuru; fatalfamilial insomnia; Gerstmann-Straussler-Scheinker syndrome; bovinespongiform encephalopathy; scrapie; feline spongiform encephalopathy;chronic wasting disease; and transmissible mink encephalopathy.
 60. Amethod of obtaining an antibody, comprising immunising an animal with anantigen, wherein the antigen comprises a peptide that comprises afragment of a prion protein or of an analogue of a prion protein,obtaining antibodies from the animal and identifying antibodies thatbind to prion, wherein the prion comprises a PrP^(Sc) prion dimer thatis infectious in animals.
 61. A method according to claim 60, whereinthe antigen comprises a carrier covalently linked to the peptide,optionally via a linker.
 62. A method according to claim 61, furthercomprising sensitising the animal to the carrier.
 63. A method accordingto claim 62, wherein sensitising the animal to the carrier comprisingadministering a priming antigen that stimulates an immune response tothe carrier.
 64. A method according to claim 63, wherein the carriercomprises a heat shock protein.
 65. A method according to claim 63,wherein the carrier is a Mycobacterial protein and the priming antigenis administered by administering Bacillus Calmette-Guerin vaccine.
 66. Amethod according to claim 60, wherein the peptide is in a cyclic form.67. A method according to claim 60, wherein the antigen comprises acomposite of repeats of the peptide.
 68. A method according to claim 67,wherein the antigen comprises a linear or cyclic dimer of the peptide.69. An antigen comprising a fragment of at least 7 amino acids of aprion protein, wherein the antigen is capable of stimulating productionof an antibody that binds to a prion comprising a PrP^(Sc) prion dimerthat is infectious in animals.
 70. An antigen according to claim 69,wherein the fragment is a cyclic fragment of the prion protein.
 71. Anantigen according to claim 69, wherein the fragment is a linear fragmentof the prion protein.
 72. An antigen according to claim 69, wherein theantigen comprises repeats of said fragment.
 73. An antigen according toclaim 72, wherein the antigen comprises a dimer of said fragment.
 74. Anantigen according to claim 69, wherein the fragment comprises any one ofSEQ ID NO:s 1 to
 8. 75. A conjugate, for stimulating production of anantibody, comprising a carrier linked to an antigen according to claim69.
 76. A method of immunizing an animal against TSE infection,comprising administering an antigen according to claim 69, to theanimal.
 77. A method according to claim 76, wherein the antigen isadministered subsequently to or simultaneously with a priming antigenthat stimulates a response to the antigen.